STRUCTURAL STUDIES OF MULTICOMPONENT BACTERIAL MONOOXYGENASES

多组分细菌单加氧酶的结构研究

• 批准号: 8169251
• 负责人: Stephen J. Lippard
• 金额: $ 0.35万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169251
• 关键词: Characteristics; Complement; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Set; Enzymes; Family; Funding; Grant; Hydrocarbons; Institution; Metals; Methane hydroxylase; Mixed Function Oxygenases; Phenol 2-monooxygenase; Protein Binding; Pseudomonas; Publications; Reporting; Research; Research Personnel; Resolution; Resources; Role; Site; Site-Directed Mutagenesis; Source; Structure; United States National Institutes of Health; Work; carboxylate; dimer; divalent metal; genetic regulatory protein; improved; mutant; reconstitution; toluene 2-xylene monooxygenase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacterial multicomponent monooxygenases are a family of enzymes that utilize a carboxylate-bridged diiron center to hydroxyalate a variety of hydrocarbon substrates. Essential for this activity is the formation of a complex between the hydroxylase and regulatory protein. The structure of the Pseudomonas sp. OX1 phenol hydroxylase, PHH-PHM complex was determined to 2.3 ¿ resolution. The regulatory protein binds on helices A, E, and F of the hydroxylase alpha subunit at the dimer interface 12 ¿ above the diiron center. Although the metal center resembled the structure of mixed-valent methane monooxygenase, significant structural changes were observed in helices E and F that were different than the configuration typically observed in the uncomplexed form of the hydroxylase. These changes have implications for substrate activation and substrate selectivity, the significance of which is currently being explored. Since determining this structure, structural work has focused on obtaining higher resolution data on the PHH-PHM complex with improved regulatory protein occupancy, and crystallographic characterization of various mutants of PHH and toluene/o-xylene monooxygenase hydroxylase from Pseudomonas sp. OX1. Higher resolution and structures of the complex with improved regulatory protein occupancy could confirm mechanistic conclusions and speculations about the structural effects of regulatory protein binding proposed upon analysis of the initial PHH-PHM data. Similarly, a structure of the hydroxylase in the absence of the regulatory protein may reveal critical, mechanistically relevant characteristics of the hydroxylase structure while also providing a platform to further investigate the structural effects of regulatory protein binding on the hydroxylase through comparison with the PHH-PHM structure. Diffraction data from these projects are only preliminary and require improvement and analysis before they may be reported. In effort to obtain a diferric or analogous structure of PHH or PHH-PHM, dithionite soaked divalent metal reconstituted and crystals of both species were pursued but not yet successfully obtained such to yield a quality diffraction data set for structure determination. Mechanistic data on mutant forms of ToMOH (T201X) and PHH (N204X) in which conserved residues near the diiron site were varied by site-directed-mutagenesis indicate roles for these residues, so the mutants were crystallized and their diffraction data collected to yield structural information that complements the mechanistic studies. This structural information is presently being analyzed and prepared for publication in conjuction with the relevant mechanistic studies and their results.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 细菌多组分单加氧酶是利用羧酸桥联铁中心对多种烃底物进行羟化的酶家族。该活性的关键是羟化酶和调节蛋白之间形成复合物。假单胞菌属的结构。 OX1 苯酚羟化酶、PHH-PHM 复合物测定分辨率为 2.3 ¿调节蛋白结合在二铁中心上方二聚体界面 12 处的羟化酶 α 亚基的螺旋 A、E 和 F 上。尽管金属中心类似于混合价甲烷单加氧酶的结构，但在螺旋E和F中观察到显着的结构变化，这与在非复合形式的羟化酶中通常观察到的构型不同。这些变化对底物活化和底物选择性具有影响，目前正在探索其重要性。自从确定了这种结构以来，结构工作的重点是获得 PHH-PHM 复合物的更高分辨率数据，以及改善的调节蛋白占据率，以及来自假单胞菌属的 PHH 和甲苯/邻二甲苯单加氧酶羟化酶的各种突变体的晶体学表征。 OX1。具有改善的调节蛋白占据率的复合物的更高分辨率和结构可以证实在分析初始 PHH-PHM 数据时提出的关于调节蛋白结合的结构效应的机械结论和推测。同样，在缺乏调节蛋白的情况下，羟化酶的结构可以揭示羟化酶结构的关键的、机械相关的特征，同时还提供一个平台，通过与 PHH-PHM 结构进行比较，进一步研究调节蛋白结合对羟化酶的结构影响。这些项目的衍射数据只是初步的，需要改进和分析才能报告。为了获得 PHH 或 PHH-PHM 的二价铁或类似结构，对连二亚硫酸盐浸泡的二价金属进行了重构，并得到了两种物质的晶体，但尚未成功获得，从而产生用于结构测定的高质量衍射数据集。 ToMOH (T201X) 和 PHH (N204X) 突变体形式的机制数据（其中二铁位点附近的保守残基通过定点诱变而改变）表明了这些残基的作用，因此突变体被结晶并收集它们的衍射数据，以产生补充机制研究的结构信息。目前正在分析该结构信息，并准备与相关机制研究及其结果一起发表。